Sulfatase enzymes, which catalyze the hydrolysis of sulfate esters, alter many substrates which have key functions in development, signaling, degradation, pathogenesis, and disease. The expression levels of the recently characterized sulfatases HSulf-1 and HSulf-2 are increased in certain cancers (e.g. breast, pancreas, colon), making early diagnosis of cancer achievable by the quantification and visualization of sulfatase activity in tumor cells. Sensitive and selective detection necessitates the design of high affinity fluorescent probes for the extracellular sulfatases HSulf-1 and HSulf-2. This proposal describes the design of fluorophores which should undergo significant alterations in quantum yield or emission after hydrolysis by extracellular sulfatases. Because one enzyme can hydrolyze many probes, the sulfatase-activated fluorescent signal will be amplified, enabling sensitive detection of abnormal, cancer-associated activity. Fluorophores with red or near-infrared emission will be targeted to enable deeper tissue penetration and to reduce tissue autofluorescence. Distinct hydrolyzable fluorophore scaffolds will be evaluated, including sulfated dimethylacridinones, resorufins, perylene diones, and luminogenic coumarin-lanthanide conjugates. The photochemical and kinetic properties will be determined for each probe in vitro, in cancer cell lines, and in mouse tumor models. Although it is known that sulfatases can hydrolyze diverse aryl sulfates, slight modifications to the dye scaffolds (e.g., fluorination) will be examined to improve the binding affinity or fluorescent properties. The probe-enzyme affinity will be further enhanced by conjugation to anionic molecules identified by screening libraries of DMA aptamers, peptides, or peptoids. In summary, the sulfatase activated probes will enable sensitive and selective fluorescence imaging of sulfatase activity inside living animals for the diagnosis of cancer. ***Sulfatases have elevated levels in certain cancers, including breast, pancreatic, and colon cancer. Through their activity, these enzymes may be controlling the severity of cancers by influencing interactions at the cell surface. The described probes are activated by extracellular sulfatases to give a distinct fluorescent signal at cancer cells. The probes are designed to enable early and accurate cancer diagnosis.